Clonig and Sequencing og Rice Prolamin Genes

• Main Authors: Chen , Jyh - Miin, 陳志敏
• Other Authors: Chen , Ching - San
• Format: Others
• Language: zh-TW
• Published: 1993
• Online Access: http://ndltd.ncl.edu.tw/handle/50712252245280431939

碩士 === 國立臺灣大學 === 農業化學系 === 81 === Rice prolamin is a group of storage proteins encoded by a complex mutigene family. These proteins have molecular weight in the range of 12 to 17 kDa. in order to investigate the expression and regulation of rice prolamin genes , a prolamin cDNA clone pS23 was used as a probe to screen a rice lambda EMBL 3 genomic library . Five positive plaques were isolated and phage DNA from one of the plaques, lambda G601, was purified and digested with Sal I. A Sal I fragment about 2.6 kb was found to hybridize to pS23 probe in Southern blotting . The Sal I fragment was subcloned into pBluescript SK II vecter to create recombinant plasmids. Two clones, pJM3 and pJM4 , were isolated from the recombinant plasmids . DNA mapping indicated that the inserts in these two clones had different orientation. Both pJM3 and pJM4 plasmids were cleved with Xho I and Kpn I , serially deleded by exonuclease III, S1 nuclease digestion , religated and tranformed into XL1-Blue . This clustered set of overlapping DNA inserts was sequencd by dideoxynucletide chain termination method. 1836 nucleotide sequences of the inserts of both pJM3 and pJM4 were determined . A 303 bp of pJM4 edcodes a 11 kDa prolamin. This gene was named RP3.The PR3 gene contains a single open reading frame encoding 101 amino acids , 1046 bp of 5'' flanking reagion, and 487 bp of 3'' flanking reagion:No intron are present. Rice prolamin cDNA are divided into three classes , namely classes I, II,IIIbased on the nucleotide sequence homology of the coding sequences. RP3 showed high homology with classes I cDNAs,pX24 and pS23,but low homology with class III cDNA, pS18.